Synergistic effects of endothelial and dental pulp stem cells in-vitro

Abstract

OBJECTIVES: To investigate the synergistic effects of dental pulp stem cells (DPSCs) and endothelial cells (ECs) on osteo/odontogenic differentiation and angiogenic potentials in vitro. METHODS: Different ratios of DPSCs and ECs were co-cultured in optimized medium. The 70% confluent co-cultures were incubated in osteo/odontogenic differentiation medium containing 10nM dexamethasone, 10mM β-glycerophosphate, 50 mg/mL ascorbate phosphate and 10nM 1, 25 dihydroxyvitamin D3 up to 3 weeks. The levels of alkaline phosphatase (ALP), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) genes and Alizarin Red staining for mineralization were analyzed. Tubular network formation on Matrigel and the gene expression levels of CD117, VEGF, CD34 and Flk-1 were determined to analyze angiogenesis. All the experiments were conducted in triplicate using DPSCs from three different donors. The data were statistically analysed using one way ANOVA. RESULTS: The level of ALP in DPSCs:ECs co-cultures was greater than that in DPSCs cultures alone. 1:1 DPSCs-ECs co-cultures at all time points showed a significantly higher ALP activity than that of DPSCs cultures. Alizarin Red staining and quantification assay revealed higher amount of calcification in the 1:1 and 1:5 co-cultures, compared to other cultures (p<0.01). With reference to DPSCs cultures, the increased expression levels of ALP, BSP and DSPP genes in co-cultures confirmed the synergistic effects of DPSCs and ECs on promotion of osteo/odontogenic differentiation. Addition of DPSCs stabilized pre-existing vessel-like structures formed by ECs and increased their longevity. The increased levels of angiogenic markers in co-cultures further confirmed the synergistic effects on angiogenesis. CONCLUSION: This study shows that co-culture of DPSCs and ECs may enhance the osteo/odontogenic differentiation and angiogenic potentials. (Supported by Hong Kong Research Grants Council (GRF) to CZ).